OBJECTIVE To produce and characterize monoclonal antibodies against the rhesus placental MHC class I molecule, Mamu-AG. RESULTS Research on placental MHC class I molecule expression has been hampered by a lack of specific antibodies to evaluate protein distribution and function. We utilized a subtractive immunization approach to produce monoclonal antibodies against the rhesus placental class I molecule Mamu-AG. Balb/C mice were immunized with the human 721.221 BLCL cell line expressing the polymorphic MHC class I molecule Mamu-A04. Mice were identified with a positive immune response by flow cytometry of serum. Four positive mice were treated with three rounds of cyclophosphamide to deplete cells of the response to the BLCLs. Mice were then immunized with BLCLs expressing Mamu-AG to generate a specific immune response against this molecule. We screened 200 hybridomas and identified two that 2 that recognized Mamu-AG but not Mamu-A04. Of these two, one clone recognized rhesus peripheral blood lymphocytes, indicating that it was not monospecific for Mamu-AG. The other antibody 25D3 recognized trophoblas ts, BLCLs expressing Mamu-AG, but not peripheral blood lymphocytes or BLCLs expressing a variety of rhesus classical MHC class I molecules. We used this antibody to evaluate Mamu-AG protein distribution at the maternal-fetal interface. Immunostaining was localized to rhesus placental trophoblasts but not to placental stromal or maternal uterine cells, verifying its noncross-reactivity with classical MHC class I molecules of maternal or fetal origin. FUTURE DIRECTIONS We will use this antibody to confirm lack of protein expression in nonplacental tissues in the rhesus, which express low levels of Mamu-AG mRNA. The antibodies will then be critical to investigate the physiological role of placental nonclassical MHC class I molecules both in vivo and in vitro. KEY WORDS placenta, major histocompatibility complex, maternal-fetal immune tolerance FUNDING HD34215, HD26458